Method of production of nanoparticle and nanoparticle produced by the method of production

ABSTRACT

The method of the production of a nanoparticle of the present invention includes a step of forming a nanoparticle including a compound of ametal ion in a cavity part of aprotein, in a solution containing the protein having the cavity part therein, the metal ion, and a carbonate ion and/or a hydrogen carbonate ion. Examples of the aforementioned compound include e.g., a hydroxide. The aforementioned metal ion is preferably any one of a nickel ion (Ni 2+ ), a chromium ion (Cr 2+ ) or a copper ion (Cu 2+ ). According to the aforementioned method, nanoparticles having a uniform particle diameter can be produced.

[0001] This is a continuation application under 35 U.S.C. 111(a) ofpending prior International Application No. PCT/JP03/11810, filed onSep. 17, 2003.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method of the production of ananoparticle in which a cavity of a protein is utilized, a nanoparticleproduced by the method of the production, and a nanoparticle-proteincomplex generated in the production step of the method of theproduction.

[0004] 2. Description of the Related Art

[0005] Mainstream of the development of functional materials implementedso far involves exploration and synthesis of novel compounds which allowperforming a desired function. However, in recent years, it has beendesired to allow performing new functions which can not be achieved in abulk state through producing nanoparticles obtained by fine division ofa substance into a nanometer size. In particular, production ofnanoparticles of semiconductors or inorganic materials including a metalcompound as a center has been strongly desired for the purpose ofproducing quantum effect devices which draw the attention in recentyears.

[0006] Methods of the production of nanoparticles which have beenconventionally carried out involve physical grinding methods, chemicalsynthesis methods and the like. For example, the physical grindingmethods have been widely used in order to obtain starting materials uponbaking of ceramics. In addition, examples of the chemical synthesismethod include methods in which gold nanoparticles are produced throughreducing chloroauric acid among long chain organic compounds. The longchain organic compound in this process inhibits the growing of a goldparticle to an enormous size.

[0007] Furthermore, there exist methods in which a complex between anorganic compound and a nanoparticle is generated followed by a chemicalreaction to result in uniform particles. As an application of thismethod, there also exists a method in which gold nanoparticles areobtained having a self-assembled monomolecular membrane (SAM membrane)formed on their surfaces through fixing a gold atom on a material forforming a SAM membrane, and assembling the material such that the goldatom becomes the center. Moreover, a method is also executed in which amicelle including a material which forms a nanoparticle is produced, andnanoparticles are produced using a chemical reaction in the micelle.

[0008] In order to produce a quantum effect device, it is essential toobtain nanoparticles having the identical diameter. Energy level whichcan be attained by an electron in a nanoparticle greatly variesdepending upon the diameter in the order of several nanometers. Hence,to give a constant diameter is important in applying nanoparticleshaving a quantum effect to an electronic circuit.

[0009] However, it is difficult to obtain nanoparticles having a uniformdiameter by the conventional methods as described above. In the physicalgrinding method, for example, it is originally difficult to make theparticle diameter smaller than the micron size, and even though the sizecould approximate the order of nanometer, no mechanism has beenestablished to accomplish a constant diameter. Hence, great spread ofthe diameter of thus resulting nanoparticles is inevitably generated. Inaddition, in the chemical synthesis method, the great spread of thediameter of the resulting nanoparticles is also generated inevitablybecause a chemical reaction is utilized therein. Further, it is alsodisadvantageous in respect of the required time period and cost.

[0010] On the other hand, in an attempt to apply biotechnology to otherfiled, there exist investigations in which nanoparticles having uniformsize in the order of nano are intended to be produced through renderingthe incorporation of a metal or a metal compound into apoferritin thatis a protein having a function to hold a metal compound. Investigationshave been carried out so that any of various kinds of metals or metalcompounds is introduced into apoferritin in compliance with the use ofthe nanoparticle.

[0011] Apoferritin is now explained below. Apoferritin is a proteinwhich is present widespread in an animate nature, and plays a role toregulate the amount of iron which is an essential trace element in aliving body. A complex of iron or an iron compound with apoferritin isreferred to as ferritin. Since iron is deleterious to a living body whenit is present in an excessive amount in the body, excess iron is storedin the body in the form of ferritin. Furthermore, ferritin returns toapoferritin through releasing an iron ion as needed.

[0012]FIG. 1 is a schematic view illustrating the structure ofapoferritin. As shown in FIG. 1, apoferritin 1 is a spherical proteinhaving the molecular weight of about 460,000 with 24 monomer subunits,which are formed from a single polypeptide chain, being assembled vianoncovalent bonds, having the diameter of about 12 nm, and exhibitshigher thermostability and pH stability in comparison with generalproteins. There is a cavity-like holding part 4 having the diameter ofabout 6 nm in the center of apoferritin 1, and the outside and theholding part 4 are connected via a channel 3. For example, when abivalent iron ion is incorporated into apoferritin 1, the iron ionenters from the channel 3, and reaches to the holding part 4 after beingoxidized in a place which is present within a part of the subunits andis referred to as a ferrooxidase center (iron oxidation active center).The iron ion is thereafter concentrated in a negative charge region onthe inner surface of the holding part 4. Then, the iron atoms assembleby 3000 to 4000, and held in the holding part 4 in the form of aferrihalide (5Fe₂O₃.9H₂O) crystal. Diameter of the nanoparticle, whichwas held in the holding part 4, comprising the metal atom is nearlyequal to the diameter of the holding part 4, which is about 6 nm.

[0013] Using this apoferritin, nanoparticle-apoferritin complexes havebeen generated in which a metal or a metal compound other than iron isartificially carried.

[0014] Introduction of a metal or a metal compound such as manganese(see, P. Mackle, 1993, J. Amer. Chem. Soc. 115, pp. 8471-8472; and F. C.Meldrum et al., 1995, J. Inorg. Biochem. 58, pp. 59-68), uranium (see,J. F. Hainfeld, 1992, Proc. Natl. Acad. Sci. USA, 89, pp. 11064-11068),beryllium (see, D. J. Price, 1983, J. Biol. Chem. 258, pp. 10873-10880),aluminum (see, J. Fleming, 1987, Proc. Natl. Acad. Sci. USA, 84, pp.7866-7870), and zinc (D. Price and J. G. Joshi, Proc. Natl. Acad. Sci.USA, 1982, 79, pp. 3116-3119) into apoferritin has been reported so far.The diameter of the nanoparticles including any one of these metals ormetal compounds is also nearly equal to the diameter of the holding part4 of apoferritin, which is about 6 nm.

[0015] Summary of the process in which nanoparticles including an ironatom are formed in apoferritin in the natural world is as follows.

[0016] On the surface of the channel 3 which connects between theoutside and inside of apoferritin 1 (see, FIG. 1) are exposed aminoacids having a negative charge under a condition of pH of 7-8, thus iron(II) ions having a positive charge are incorporated into the channel 3by an electrostatic interaction. On the inner surface of the holdingpart 4 of apoferritin 1 are exposed a lot of glutamic acid residueswhich are amino acid residues having a negative charge at pH 7-8,similarly to the inner surface of the channel 3, and the iron (II) ionsincorporated from the channel 3 are oxidized at the ferroxidase center,followed by introduction to the internal holding part 4. Then, the ironions are concentrated by an electrostatic interaction, and the coreformation of a ferrihalide (5Fe₂O₃.9H₂O) crystal is caused.

[0017] Thereafter, a core which comprises iron oxide is grown throughadherence of the iron ions, which are sequentially incorporated, to thecrystal core, and accordingly, a nanoparticle having the particlediameter of 6 nm is formed in the holding part 4. Summary of theformation of a nanoparticle including iron oxide as well as theincorporation of the iron ions is as set forth above.

[0018] Although a mechanism of the incorporation of iron ions intoapoferritin was described hereinabove, it is believed that other metalions, which were reported hitherto as candidates for the introduction,also involve an approximately similar mechanism to that for the ironion. However, metal ions which can be incorporated in the cavity part bythe method described above were limited to particular ones.

SUMMARY OF THE INVENTION

[0019] An object of the present invention is to provide a method of theproduction a nanoparticle in which a cavity of a protein is utilized,which enables to obtain also nanoparticles having a uniform particlediameter including a metal ion such as nickel ion (Ni²⁺), chromium ion(Cr²⁺), copper ion (Cu²⁺) or the like which has not been hithertoreported on the formation of a nanoparticle in a cavity part of aprotein.

[0020] The present invention is directed to a method of the productionof a nanoparticle which comprises a step of forming a nanoparticle,which includes compound of a metal ion in a cavity part of a protein, ina solution containing the protein having the cavity part therein, themetal ion, and a carbonate ion and/or a hydrogen carbonate ion.

[0021] Examples of the aforementioned compound include e.g., ahydroxide.

[0022] The aforementioned metal ion is preferably any one of a nickelion (Ni²⁺), a chromium ion (Cr²⁺) or a copper ion (Cu²⁺).

[0023] The pH of the aforementioned solution is preferably approximatelyequal to a precipitation point of a hydroxide of the aforementionedmetal ion.

[0024] When a nickel ion is used as the aforementioned metal ion, the pHof the aforementioned solution is preferably 8 or greater and 9 or less.

[0025] In addition, when a nickel ion is used as the aforementionedmetal ion, the aforementioned solution preferably includes an ammoniumion, and in this instance, the pH of the aforementioned solution ispreferably greater than 8.3 and equal to or less than 8.65.

[0026] For example, the aforementioned protein is at least one ofapoferritin, Dps protein, CCMV protein, TMV protein or a heat shockprotein.

[0027] For example, the aforementioned solution contains a carbonate ionand/or a hydrogen carbonate ion produced by bubbling carbon dioxidethereto.

[0028] After forming the aforementioned nanoparticle, the method mayfurther comprise a step of eliminating the protein by a heat treatment.

[0029] In addition, the present invention concerns a nanoparticleincluding a compound of a metal ion, which is formed in a cavity part ofa protein, in a solution containing the protein having the cavity parttherein, the metal ion, and a carbonate ion and/or a hydrogen carbonateion.

[0030] Moreover, the present invention concerns a complex comprising aprotein having a cavity part therein, and a nanoparticle formed in thecavity part of the protein; the nanoparticle being a nanoparticleincluding a compound of a metal ion, which is formed in the cavity partof the protein, in a solution containing the protein, the metal ion, anda carbonate ion and/or a hydrogen carbonate ion.

[0031] These objects as well as other objects, features and advantagesof the invention will become apparent to those skilled in the art fromthe following description with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 is a schematic view illustrating the structure ofapoferritin.

[0033]FIG. 2 is a flow chart illustrating a method of the production ofa nickel compound-apoferritin complex of Embodiment 1.

[0034]FIG. 3 is a process sectional view illustrating a method of theproduction of a nonvolatile memory cell.

[0035]FIG. 4 is a process sectional view illustrating a method ofarranging and fixing dot bodies on the surface of a basal plate in a twodimensional fashion.

[0036]FIG. 5 is an explanatory view illustrating a method of arrangingand fixing complexes on the surface of a basal plate in a twodimensional fashion.

[0037]FIG. 6 is a top view illustrating a nonvolatile memory cell.

[0038]FIGS. 7A, B and C show electron micrographs illustrating states ofthe formation of the nanoparticles in Example 2.

[0039]FIGS. 8A, B, C and D are views illustrating the relationshipbetween time at each concentration of ammonium nickel sulfate, andapoferritin concentration and the yield of formation in Example 3.

[0040]FIG. 9 is a view illustrating the relationship between theconcentration of ammonium nickel sulfate and efficiency of formation inExample 3.

[0041]FIGS. 10A and B show electron micrographs illustrating states ofthe formation of the nanoparticles in Example 4.

[0042]FIG. 11A is a view illustrating the relationship between pH andyield of formation, and FIG. 11B is a view illustrating the relationshipbetween pH and efficiency of formation in Example 5.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Embodiments of the present invention are explained below withreference to accompanying drawings.

[0044] (Embodiment 1)

[0045] The method of the production of a nanoparticle according to thisembodiment is explained with reference to FIG. 1 and FIG. 2.

[0046]FIG. 2 is a flow chart illustrating a method of the production ofa complex comprising a protein including a nanoparticle of a nickelcompound therein of this embodiment (hereinafter, may be also referredto as “nickel compound-apoferritin complex”)

[0047] First, as shown in FIG. 2, in the step St1, a buffer including amixed solution of HEPES and CAPSO, an apoferritin solution, a nickelsalt solution, Milli Q water, and carbon dioxide are provided. Thebuffer, apoferritin solution, nickel salt solution and Milli Q waterprovided for use in this step should be degassed.

[0048] Next, in the step St2, carbon dioxide-bubbled water is preparedby bubbling carbon dioxide in degassed Milli Q water.

[0049] Next, in the step St3, a reaction solution is prepared by addingthe buffer, apoferritin solution and nickel salt solution to the carbondioxide-bubbled water obtained in the aforementioned step St2.Thereafter, the reaction solution is left to stand for 24 hours.

[0050] The operations for producing the nickel compound-apoferritincomplex explained hereinabove are all conducted at room temperature, orin the range of the temperature which does not result in denaturation ofapoferritin, while stirring with a stirrer.

[0051] By the aforementioned operations, the compound of a nickel ion(Ni²⁺) is introduced into a holding part 4 of apoferritin 1 to form anickel compound-apoferritin complex.

[0052] Proteins including apoferritin are produced on the basis ofinformation of a DNA, and are readily reproduced in a large number byany known method. Further, it is well known that proteins reproducedfrom the identical DNA in a large number have the same structure in anaccuracy of the angstrom level. Therefore, the cavity like holding parts4 carried by apoferritin used in this embodiment all have the same sizeand shape.

[0053] Therefore, when nanoparticles are produced in proteins as in thisembodiment, the diameter of the nanoparticle is defined by the proteinto result in nanoparticles having a uniform particle diameter. Forexample, in accordance with this embodiment, the diameter of thenanoparticle becomes 6 nm which is almost the same as the diameter ofthe holding part of apoferritin.

[0054] Although apoferritin was used as a protein in this embodiment,any protein constituted from a single subunit or any protein constitutedfrom multiple subunits may be used in stead of apoferritin as long asthe protein has a cavity part. Further, the protein is not limited toone with a cavity part having a spherical form, but may be any one witha cavity part having a rod shape, a ring shape or the like. For example,Dps protein, viral protein, a heat shock protein or the like may beused. When Dps protein (a spherical shell-like protein having thediameter of 9 nm, and having a holding part therein with the diameter of4 nm) is used, nanoparticles having the diameter of 4 nm can beproduced. Examples of the viral protein include for example, CPMV, CCMV,HSV, Rotavirus, Reovirus, LA-1, Polyoma, CaMV, HPV, Ross River, SpV-4,pX174, FHV, HRV-14, Polio and the like. Preferably, a viral protein suchas CPMV or CCMV may be used. According to the method of this embodiment,formation of nanoparticles are rendered in compliance with the shape andsize of the cavity part of the protein used. Nanoparticle herein refersto a particle having a major axis of 50 nm or less, and having the sizeat least allowing for stable presence as a particle. An exemplarynanoparticle corresponds to a particle having a major axis of 1 nm to 50nm.

[0055] Moreover, although the production of a nanoparticle composed of anickel compound is explained in this embodiment, the method of thisembodiment can be applied to also in the case where a nanoparticlecomposed of a compound including a metal atom such as chromium or copperis produced. Metal compounds which may constitute the nanoparticleformed by the method of this embodiment vary depending on the addedmetal ion or the like, however, they may be for example, a hydroxideand/or an oxide.

[0056] When nanoparticles composed of a chromium compound are produced,a chromium salt solution is mixed in the reaction solution in stead ofthe nickel salt solution used in this embodiment. Other conditions areas set forth in this embodiment. When nanoparticles composed of a coppercompound are produced, a copper salt solution is mixed in the reactionsolution in stead of the nickel salt solution used in this embodiment.Other conditions are as set forth in this embodiment.

[0057] The presence of a hydrogen carbonate ion (HCO³⁻) and/or carbonateion (CO₃ ²⁻) in the reaction solution is important for the formation ofa nanoparticle in this embodiment. Therefore, bubbling of carbon dioxidein degassed Milli Q water is executed in the step St2 in thisembodiment, however, this step is not limited thereto but a process inwhich a hydrogen carbonate ion (HCO³⁻) and/or carbonate ion (CO₃ ²⁻) isadded to the reaction solution may be employed. For example, a carbonatesuch as sodium carbonate (Na₂CO₃) or sodium bicarbonate (NaHCO₃) or thelike may be used through dissolving in Milli Q water.

[0058] In accordance with this embodiment, the buffer for use is a mixedsolution of HEPES and CAPSO. However, means for adjusting the pH is notlimited thereto, but any means may be employed. For example, a buffercontaining one kind of a buffering agent may be used, or a mixedsolution of multiple kinds of buffering agents may be also used as abuffer. In particular, using a mixed solution of multiple kinds ofbuffering agents as a buffer is more advantageous than using a buffercontaining one kind of a buffering agent, because the pH of the reactionsolution can be readily adjusted. Accordingly, it is preferred that amixed solution of multiple kinds of buffering agents is used as abuffer.

[0059] In this embodiment, ammonium nickel sulfate (NiNH₃(SO₄)) is usedas the nickel salt solution, but not limited thereto. For example,nickel sulfate (Ni(SO₄)), nickel nitrate (Ni (NO₃)₂) or nickel chloride(NiCl₂) may be also used. In this embodiment, it is desired that anammonium ion (NH₄ ⁺) is included in the reaction solution in light ofthe reaction yield. When an ammonium metal salt solution is used as ametal salt solution, the reaction solution shall contain an ammonium ionwithout adding any other component to the reaction solution.

[0060] In accordance with this embodiment, time period for leaving tostand after preparing the reaction solution is set to be 24 hours,however the time period is not limited thereto, but it is desirable toselect the most appropriate time period to comply with the constitution,pH and the like of each reaction solution.

[0061] More specific conditions and the like of the reaction solution inconnection with the method for the production of a nanoparticledemonstrated in this embodiment are described below in the section ofExamples.

[0062] (Embodiment 2)

[0063] In this embodiment, a nonvolatile memory cell is explained, whichincludes dot bodies composed of nanoparticles produced by utilizingnickel compound-apoferritin complexes produced in the embodiment 1described above, as a floating gate.

[0064] FIGS. 3(a) to (d) are process sectional views illustrating amethod of the production of a nonvolatile memory cell of thisembodiment.

[0065] First, in the step depicted in FIG. 3(a), on a type p Si basalplate 101 is formed a chip separating oxide membrane 102 surrounding theactive region by a LOCOS method, and thereafter a gate oxide membrane103 which functions as a tunnel insulation membrane is formed on thebasal plate by a thermal oxidation method. Then, dot bodies 104 composedof a nanoparticle of nickel having the diameter of approximately 6 nmare formed on the basal plate. Process for forming the dot bodies 104 onthe basal plate is explained later.

[0066] Next, in the step depicted in FIG. 3(b), on the basal plate isdeposited a SiO₂ membrane into which the dot bodies 104 are embedded bya sputter method or a CVD method.

[0067] Next, in the step depicted in FIG. 3(c), an Al membrane isdeposited on the basal plate. Subsequently, a silicon oxide membrane 105which shall be an insulation membrane between electrodes and an Alelectrode 106 which shall be a regulation gate electrode are formed bypatterning of the SiO₂ membrane and Al membrane using a photo resistmask Pr1. Upon this manipulation, a part of the gate oxide membrane 103without being covered by the photo resist mask Prl is eliminated,therefore, the dot bodies 104 thereon are concurrently eliminated.Thereafter, first and second diffusion layers 107 a and 107 b of type nare formed through carrying out the infusion of an impure ion, with thephoto resist mask and the Al electrode 106 as masks.

[0068] Then, in the step depicted in FIG. 3(d), formation of aninsulation membrane 108 between layers; opening of a contact hole 109 ofthe insulation membrane 108 between layers; formation of a tungsten plug110 by embedding tungsten into a contact hole 109; and formation offirst and second aluminum wirings 111 a and 111 b are carried out by anyknown method.

[0069] Although an Si basal plate of type p was used as a basal plate inthis embodiment, also an Si basal plate of type n may be used, oralternatively, any basal plate which is constituted from othersemiconductor such as a compound semiconductor including GaAs may bealso used.

[0070] Next, a process for forming dot bodies 104 on the basal plate inthe step depicted in FIG. 3(a) is explained below with reference to FIG.4 and FIG. 5. However, the process for forming dot bodies 104 on thebasal plate is not limited to the process as explained below, but anyother known process can be also applied. First, in the step depicted inFIG. 4(a), nickel compound-apoferritin complexes (hereinafter,abbreviated as “complex” in this embodiment) 150 obtained in theaforementioned embodiment 1 are provided, and these complexes 150 arearranged on the surface of a basal plate 130. Accordingly, a complexmembrane having the complexes 150 arranged on the surface of the basalplate 130 at high density and with high accuracy is formed. The basalplate 130 herein refers to that formed on an Si basal plate 101 of typep in the step depicted in FIG. 3(a) by a LOCOS method through forming achip separating oxide membrane 102 surrounding the active region, andthereafter forming a gate oxide membrane 103 which functions as a tunnelinsulation membrane on the basal plate by a thermal oxidation method.Similar matter can be applied in the following explanation.

[0071] Next, in the step depicted in FIG. 4(b), dot bodies 104 areformed on the basal plate 130 by eliminating the protein molecules 140in the complexes 150 to leave the nanoparticles 104 a of the nickelcompounds alone.

[0072] In the step depicted in FIG. 4(a), the process for arranging thecomplexes 150 on the surface of the basal plate 130 at high density andwith high accuracy, in other words, for aligning and fixing in a twodimensional fashion on the surface of the basal plate 130 is nowexplained. In this embodiment, a process described in JP-A No. 11-45990is adopted. The process is explained below with reference to FIG. 5.

[0073] First, in the step depicted in FIG. 5(a), a liquid 160 (thenickel compound-apoferritin complexes dispersed in a mixed solution ofan equal amount of a phosphate buffer solution having the concentrationof 40 mM and pH of 5.3 and an aqueous sodium chloride solution havingthe concentration of 40 mM) including complexes 150 dispersed therein isprovided.

[0074] Subsequently, in the step depicted in FIG. 5(b), PBLH(Poly-1-Benzil-L-Histidine) is gently developed on the surface of theliquid 160 with a syringe or the like. A polypeptide membrane 170 whichis composed of PBLH is thereby formed on the surface of the liquid 160.Thereafter, the pH of the liquid 160 is adjusted.

[0075] Next, in the step depicted in FIG. 5(c), the complexes 150 areadhered onto the polypeptide membrane 170 in a time dependent manner toyield two dimensional crystals of the complexes 150. This is caused bythe positively charged polypeptide membrane 170, contrary to thenegatively charged complexes 150.

[0076] Next, in the step depicted in FIG. 5(d), the basal plate 130 isplaced (floated) on the polypeptide membrane 170 to allow adhesion ofthe polypeptide membrane 170 onto the basal plate 130.

[0077] Next, by recovering the basal plate 130 in the step depicted inFIG. 5(e), the basal plate 130 having two dimensional crystals of thecomplexes 150 adhered via the polypeptide membrane 170 can be obtained.

[0078] Next, the step depicted in FIG. 4(b) is explained in more detail.Since a protein molecule is generally weak against heat, elimination ofthe protein molecule 140 in the complex 150 is carried out by a heattreatment. For example, by standing still in an inert gas such asnitrogen or the like at 400-500° C. for about 1 hour, the proteinmolecule 140 and the polypeptide membrane 170 are burnt out, and thusnickel nanoparticles 104 a remain on the basal plate 130 in a twodimensional fashion as dot bodies 104 which are regularly aligned athigh density and with high accuracy.

[0079] Accordingly, the dot bodies 104 which are aligned at high densityand with high accuracy can be formed by rendering the nickelnanoparticles 104 a that were held by the complexes 150 appear on thebasal plate 130 in a two dimensional fashion, as shown in FIG. 4(b).Heat treatment was employed as the process for eliminating the proteinmolecules 140 in this embodiment, however, the process is not limitedthereto but may be executed by deleting through the decomposition of theprotein using for example, a proteolytic enzyme, a chemical substance orthe like.

[0080] As shown in FIG. 3(d), the memory cell 100 of this embodiment isequipped with the Al electrode 106 which functions as a regulation gateand a MOS transistor (memory cell transistor) composed of the first andsecond diffusion layers 107 a and 107 b of type n which function as asource or a drain, and is a nonvolatile memory cell in which alterationof threshold potential of the aforementioned memory transistor isutilized depending upon the amount of the charge accumulated in the dotbodies 104 which function as a floating gate.

[0081] A function as a memory which memorizes two values can be achievedaccording to this nonvolatile memory cell. In addition, nickel, chromiumand copper are different in terms of their work functions from iron,manganese, cobalt and the like, as shown in Table 1. The work functionspresented in Table 1 are cited from H. B. Michaelson “Work Functions ofthe elements” Journal of Applied Physics, pp. 536-540, volume 21, 1950.TABLE 1 Metal Work function Mn 4.08 Zn 4.33 Fe 4.60 Co 4.97 Ni 5.22 Pt5.63 Cr 4.50 Cu 4.50

[0082] Consequently, control of the accumulation of electrons is enabledby utilizing these nanoparticles in combination through the use ofdifferences in likelihood of accumulation of the electrons therein.Therefore, versatility of design of the quantum effect device can beachieved.

[0083]FIG. 6 is a top view illustrating a nonvolatile memory cell 100′having exactly the same constitution as that of the aforementionedmemory cell 100 except that multiple kinds of dot bodies 104, which areformed from compounds having different work functions with each other,are formed as a floating gate.

[0084] As is shown in FIG. 6, by forming multiple kinds of dot bodies104, which are formed from compounds having different work functionswith each other, as a floating gate, memory of multiple values that arethree values or more (four values memory in the nonvolatile memory cell100′) can be also put into practice capable of controlling not only thepresence of the charge accumulated in the dot bodies 104 but also theamount of accumulation of the charge.

[0085] Upon clearing data, an FN (Fowler-Nordheim) current via an oxidemembrane or a direct tunneling current is utilized.

[0086] Further, for writing data, an FN current via the oxide membrane,a direct tunneling current or channel hot electron (CHE) infusion isemployed.

[0087] Since the nonvolatile memory cell of this embodiment is composedof nickel nanoparticles having the particle diameter small enough toallow the floating gate to function as a quantum dot, the amount of theaccumulation of charge becomes slight. Therefore, the amount of currentupon writing and clearing can be diminished, and thus a nonvolatilememory cell with low electric power consumption can be constituted.

[0088] Additionally, according to the nonvolatile memory cell of thisembodiment, because the size of the nickel dot bodies 104 thatconstitute the floating gate is uniform, properties exhibited duringinfusion and drawing of the charge are uniform among respective nickeldot bodies 104, and thus regulation upon these manipulations can bereadily conducted.

EXAMPLE Example 1 Production of Nickel Compound-Apoferritin Complex

[0089] In this Example, each of the solutions was first prepared, i.e.,an apoferritin solution in which HEPES buffer, CAPSO buffer andcommercially available apoferritin (derived from equine spleen) weredissolved, and an ammonium nickel sulfate solution. Concentration and pHof each solution are as shown in Table 2. After preparing each solution,degassing of the HEPES buffer and CAPSO buffer was immediatelyconducted. TABLE 2 Solution (pH) Concentration HEPES buffer (pH 7.5) 500mM CAPSO buffer (pH 9.5) 500 mM Apoferritin solution  55 mg/ml Ammoniumnickel sulfate solution 200 mM

[0090] Next, Milli Q water was provided, and carbon dioxide-bubbledwater was prepared by bubbling carbon dioxide into Milli Q water for 30minutes. Immediately thereafter, a reaction solution was prepared havingthe constitution presented in Table 3 by admixing each solutionpresented in Table 2 into the carbon dioxide-bubbled water. In thisExample, reaction solutions having three different CAPSO concentrationswere prepared. Thus, reaction solutions having three kinds of pH wereprepared. TABLE 3 Solution (pH) Concentration HEPES 150 mM CAPSO 235,250, 265 mM Apoferritin 0.3 mg/ml Ammonium nickel sulfate 5 mM

[0091] In this Example, each reaction solution having the constitutionpresented in Table 3 was prepared to give the total volume of 3 ml,therefore, the amount added of carbon dioxide-bubbled water and eachsolution presented in Table 2 was as shown in Table 4. TABLE 4 Solution(concentration, pH) Concentration Carbon dioxide-bubbled water Added togive total volume of 3 ml HEPES buffer (500 mM, pH 7.5) 900 μl CAPSObuffer (500 mM, pH 9.5) 1410, 1500, 1590 μl Apoferritin solution (55mg/ml) 16.35 μl Ammonium nickel sulfate solution 75 μl

[0092] Each of the three kinds of reaction solutions obtained asdescribed above was left to stand at 23° C. for 24 hours. Thereafter,each reaction solution was centrifuged at 8000 G for 30 minutes. Eachsupernatant was collected, and the state of the supernatant wasobserved.

[0093] Next, each of thus resulting supernatants was diluted to threefold with water, stained apoferritin with 2% gold glucose, and observedwith a transmission electron microscope (TEM) with fifty thousand foldmagnification. When staining is conducted with 2% gold glucose, entryinto the holding part in apoferritin does not take place. Therefore,apoferritin having a void holding part, and a nickelcompound-apoferritin complex can be discriminated.

[0094] Observation of each supernatant with a transmission electronmicroscope revealed a large number of apoferritin including ananoparticle of a nickel compound therein, with the appearance of aprotein portion having a white colored doughnut like shape, with acentral portion which appears black. Nanoparticles of the nickelcompound had a spherical shape, with the diameter of 6 nm (standarddeviation: 1 nm) In other words, it is deemed that nanoparticles havinga uniform particle diameter could be obtained.

[0095] The results of observation as described above are presented inTable 5 as a relationship between the concentration of the CAPSO bufferand the yield of nanoparticle formation (hereinafter, may be alsoreferred to as YCF). The yield of nanoparticle formation (YCF) is aratio of apoferritin including the nanoparticle of the nickel compound(i.e., nickel compound-apoferritin complex) therein among apoferritin inthe supernatant. TABLE 5 CAPSO concentration 235 mM 250 mM 265 mM pH 8.28.3 8.4 Yield of nanoparticle formation 90% 100% 100%

[0096] On the other hand, results obtained by cases in which conditionsemployed were exactly the same except that nitrogen bubbled water andoxygen bubbled water were used in stead of the aforementioned carbondioxide-bubbled water are shown in Table 6 and Table 7, respectively.Table 6 presents the results when nitrogen bubbled water was used,whilst Table 7 presents the results obtained when oxygen bubbled waterwas used. TABLE 6 CAPSO concentration 235 mM 250 mM 265 mM pH 8.2 8.38.4 Yield of nanoparticle formation 0% 5% 20%

[0097] TABLE 7 CAPSO concentration 235 mM 250 mM 265 mM pH 8.1 8.2 8.3Yield of nanoparticle formation 0% 5% 20%

[0098] When the states of these supernatants were observed, thesupernatant of the reaction solution including CAPSO at theconcentration of 250 mM was turbid, and plenty of precipitations ofNi(OH)₂ were found in the supernatant of the reaction solution includingCAPSO at the concentration of 265 mM. Therefore, it is believed that theprecipitation point of Ni (OH)₂ in the constitution of the reactionsolution of this Example is approximately pH 8.3 (approximately pH 8.2when oxygen bubbled water was used). At the higher pH than theprecipitation point, nickel ions (Ni²⁺) rapidly precipitates in the formof Ni(OH)₂, thus, it is believed that a lot of apoferritin aggregates togive precipitates accompanied by such precipitation of Ni(OH)₂. On theother hand, it is believed that formation of nanoparticles becomesdifficult as the pH becomes lower, because the presence of enoughhydroxide ion to form nanoparticles is not ensured in the solution.Therefore, it is concluded that preferable condition for efficientlyforming nanoparticles involves the pH which is approximate to theprecipitation point or the pH which is slightly lower than theprecipitation point.

[0099] From the results shown in Tables 5 to 7, it is believed that ananoparticle is formed in the holding part of apoferritin when the pHapproximates to the precipitation point in a solution containingapoferritin because a condition which allows for the formation of acompound from a nickel ion is provided. However, it is in factexceptionally difficult to form a nanoparticle without causingaggregation and precipitation of apoferritin.

[0100] In this Example, in any of the cases where carbon dioxide-bubbledwater, nitrogen bubbled water and oxygen bubbled water were used, theaggregation and precipitation of apoferritin was found in thesupernatant of the reaction solution, which contains CAPSO at theconcentration of 265 mM, having the pH higher than the precipitationpoint. Accordingly, it is believed that recovery of the nickelcompound-apoferritin complex is difficult, and thus industrialutilization thereof is not realistic.

[0101] However, as shown in Tables 5 to 7, in the supernatant of thereaction solution including CAPSO at the concentration of 265 mM,nanoparticles were formed in the holding part of approximately 100% ofapoferritin only in instances where carbon dioxide-bubbled water wasused.

[0102] Moreover, when carbon dioxide-bubbled water was used,nanoparticles of the nickel compound were formed in the holding part ofmost apoferritin at the pH which is slightly lower than theprecipitation point (in the supernatant of the reaction solutionincluding CAPSO at the concentration of 235 mM and 250 mM), and theaggregation and precipitation of apoferritin was also suppressed.

[0103] To the contrary, in instances where nitrogen bubbled water andoxygen bubbled water were used, nanoparticles were not formed in theholding part of most apoferritin at the pH which is approximate to theprecipitation point or is slightly lower than the precipitation point(in the supernatant of the reaction solution including CAPSO at theconcentration of 235 mM and 250 mM).

[0104] That is, formation of nanoparticles in the holding part ofapoferritin while suppressing the aggregation of aggregation could beachieved only in instances where carbon dioxide-bubbled water was used.

Example 2 Test for Examining Influences of Kinds of Anion of Nickel SaltUpon Production of Nickel Compound-Apoferritin Complex

[0105] In this Example, each of the solutions was first prepared, i.e.,anapoferritin solution in which HEPES buffer, CAPSO buffer andcommercially available apoferritin (derived from equine spleen) weredissolved, and nickel salt solutions (nickel sulfate solution, nickelnitrate solution and nickel chloride solution). Concentration and pH ofeach solution are as shown in Table 8. After preparing each solution,degassing of the HEPES buffer and CAPSO buffer was immediatelyconducted. TABLE 8 Solution (pH) Concentration REPES buffer (pH 7.5) 500mM CAPSO buffer (pH 9.5) 500 mM Apoferritin solution  55 mg/ml Nickelsalt solution 200 mM

[0106] Next, Milli Q water was provided, and carbon dioxide-bubbledwater was prepared by bubbling carbon dioxide into Milli Q water for 30minutes. Immediately thereafter, a reaction solution was prepared havingthe constitution presented in Table 9 by admixing each solutionpresented in Table 8 into the carbon dioxide-bubbled water. In thisExample, reaction solutions having three different CAPSO concentrationswere prepared. Thus, reaction solutions having three kinds of pH wereprepared. TABLE 9 Solution (pH) Concentration HEPES 150 mM CAPSO 140,150, 160 mM Apoferritin 0.3 mg/ml Ammonium nickel sulfate 5 mM

[0107] In this Example, each reaction solution having the constitutionpresented in Table 9 was prepared to give the total volume of 3 ml,therefore, the amount added of carbon dioxide-bubbled water and eachsolution presented in Table 8 was as shown in Table 10. TABLE 10Solution (concentration, pH) Concentration Carbon dioxide-bubbled waterAdded to give total volume of 3 ml HEPES buffer (500 mM, pH 7.5) 900 μlCAPSO buffer (500 mM, pH 9.5) 840, 900, 960 μl Apoferritin solution (55mg/ml) 16.35 μl Nickel salt solution (200 mM) 75 μl

[0108] Each of the three kinds of reaction solutions obtained asdescribed above was left to stand at 23° C. for 24 hours. Thereafter,each reaction solution was centrifuged at 8000 G for 30 minutes. Eachsupernatant was collected, and the state of the supernatant wasobserved. Each supernatant of the reaction solution including CAPSO atthe concentration of 160 mM (pH 8.24) was slightly turbid, thus it wasbelieved that the precipitation point of Ni(OH)₂ in the constitution ofthe reaction solution of this Example was approximately pH 8.2.

[0109] Next, each of thus resulting supernatants was diluted to threefold with water, stained apoferritin with 2% gold glucose, and observedwith a transmission electron microscope (TEM) with fifty thousand foldmagnification. When staining is conducted with 2% gold glucose, entryinto the holding part in apoferritin does not take place. Therefore,apoferritin having a void holding part, and a nickelcompound-apoferritin complex can be discriminated.

[0110] Results of observation of each supernatant with a transmissionelectron microscope are illustrated in FIGS. 7A to C. As is shown inFIGS. 7A to C, apoferritin including a nanoparticle of a nickel compoundtherein, with the appearance of a protein portion having a white coloreddoughnut like shape, with a central portion which appears black, wasobserved. In any of the supernatants, nanoparticles of the nickelcompound had a spherical shape, with the diameter of 6 nm (standarddeviation: 1 nm). In other words, it is deemed that nanoparticles havinga uniform particle diameter could be obtained.

[0111] The results of observation as described above are presented inTable 11 as a relationship between the concentration of CAPSO and theyield of nanoparticle formation (YCF) versus each nickel salt used.TABLE 11 CAPSO Yield of nanoparticle formation (%) concentration Nickelsulfate Nickel nitrate Nickel chloride 140 mM (pH8.14) 40-50 30-40 50-60160 mM (pH 8.19) 100 100 100 180 mM (pH 8.24) 100 100 100

[0112] As is clear from Table 11, nanoparticles of a nickel compoundwere formed in the holding part of almost 100% apoferritin at the pH ofapproximately 8.2 which is a precipitation point of Ni(OH)₂ in theconstitution of the reaction solution of this Example. Furthermore, alsoin each supernatant of the reaction solution including CAPSO at theconcentration of 140 mM (pH 8.14), it is revealed that nanoparticles ofa nickel compound were formed in the holding part of apoferritin,although the yield of nanoparticle formation was low.

[0113] From the results of this Example and the results of theaforementioned Example 1, it is concluded that the pH which isapproximate to or is slightly lower than the precipitation point ofNi(OH)₂ is suitable for efficiently forming nanoparticles of a nickelcompound in the holding part of apoferritin.

[0114] Moreover, from the results of this Example, the yield ofnanoparticle formation was approximately the same irrespective of kindsof anion of the nickel salt. Therefore, it is revealed that anion of thenickel salt does not affect the formation of nanoparticles.

Example 3 Test for Exploring Optimal Conditions: pH; Concentration ofAmmonium Nickel Sulfate Solution; and Time Period for Leaving to Stand,Upon Production of Nickel Compound-Apoferritin Complex

[0115] In this Example, each of the solutions was first prepared, i.e.,anapoferritin solution in which HEPES buffer, CAPSO buffer andcommercially available apoferritin (derived from equine spleen) weredissolved, and an ammonium nickel sulfate solution. Concentration and pHof each solution are as shown in Table 12. After preparing eachsolution, degassing of the HEPES buffer and CAPSO buffer was immediatelyconducted. TABLE 12 Solution (pH) Concentration HEPES buffer (pH 7.5)500 mM CAPSO buffer (pH 9.5) 500 mM Apoferritin solution  55 mg/mlAmmonium nickel sulfate solution 200 mM

[0116] Next, Milli Q water was provided, and carbon dioxide-bubbledwater was prepared by bubbling carbon dioxide into Milli Q water for 30minutes. Immediately thereafter, twelve kinds of reaction solutionshaving the constitutions 1 to 12 presented in Table 13 were prepared byadmixing each solution described above into the carbon dioxide-bubbledwater such that the total volume became 3 ml. TABLE 13 Ammonium HEPESCAPSO Apoferritin nickel (mM) (mM) (mg/ml) sulfate (mM) Constitution 1150 220 0.3 2 Constitution 2 150 240 0.3 2 Constitution 3 150 260 0.3 2Constitution 4 150 220 0.3 3 Constitution 5 150 240 0.3 3 Constitution 6150 260 0.3 3 Constitution 7 150 220 0.3 5 Constitution 8 150 240 0.3 5Constitution 9 150 260 0.3 5 Constitution 10 150 220 0.3 10 Constitution11 150 240 0.3 10 Constitution 12 150 260 0.3 10

[0117] Each of the solutions having twelve kinds of constitutionsobtained as described above was left to stand at 23° C. for 0, 8, 16,24, 32, 40 and 48 hours. The pH of each solution is as shown in Table14, and the pH was not altered after leaving to stand for 48 hours.TABLE 14 pH of solution Constitution 1 8.74 Constitution 2 8.82Constitution 3 8.93 Constitution 4 8.48 Constitution 5 8.58 Constitution6 8.73 Constitution 7 8.32 Constitution 8 8.42 Constitution 9 8.5Constitution 10 8.14 Constitution 11 8.15 Constitution 12 8.16

[0118] After leaving to stand for a predetermined time period, eachreaction solution was centrifuged at 8000 G for 10 minutes. Eachsupernatant was collected, and the state of the supernatant wasobserved.

[0119] First, the concentration of apoferritin in the resultingsupernatant was determined using a protein assay kit (manufactured byBioRad).

[0120] Next, the resulting each supernatant was diluted to 3 fold withwater, stained apoferritin with 2% gold glucose, and observed with atransmission electron microscope (TEM) with fifty thousand foldmagnification. When staining is conducted with 2% gold glucose, entryinto the holding part in apoferritin does not take place. Therefore,apoferritin having a void holding part, and a nickelcompound-apoferritin complex can be discriminated. A nickelcompound-apoferritin complex presented the appearance of a proteinportion having a white colored doughnut like shape, with a centralportion including a nanoparticle of a nickel compound which appearsblack.

[0121] The results of observation above are illustrated in FIGS. 8A to Das a relationship between the time period for leaving to stand and theconcentration of apoferritin (solid curve), and as a relationshipbetween the time period for leaving to stand and the yield ofnanoparticle formation (YCF) (dashed curve). FIGS. 8A, B, C and Dillustrate the cases in which the concentration of ammonium nickelsulfate is 2, 3, 5 and 10 mM, respectively. Any of the solutionsincluding CAPSO at the concentration of 260 mM (compositions 3, 6, 9 and12) resulted in precipitation of almost all apoferritin after leaving tostand for 8 hours, with the concentration of apoferritin in thesupernatant of approximately 0. Therefore, the results obtained therebyare omitted in FIGS. 8A, B, C and D. Thus, the results illustrated inFIG. 8 are: A for samples 1 and 2; B for samples 4 and 5; C for samples7 and 8; D for samples 10 and 11.

[0122] As is clear from the results as described above, precipitationpoints of Ni(OH)₂ in the cases where the concentration of ammoniumnickel sulfate is 2, 3, 5 and 10 mM, respectively, were approximately8.82, 8.58, 8.42 and 8.15.

[0123] Next, the results of observation described above are illustratedin FIG. 9 as a relationship between the concentration of ammonium nickelsulfate and the efficiency of nanoparticle formation (ECF) (solidcurve), and as a relationship between the concentration of ammoniumnickel sulfate and the pH of precipitation point (dashed curve). Theefficiency of nanoparticle formation (ECF) is a ratio of apoferritinincluding the nanoparticle therein (i.e., nickel compound-apoferritincomplex) in the supernatant among apoferritin which was added initially(when the solution was prepared).

[0124] From the results illustrated in FIG. 9, the efficiency ofnanoparticle formation (ECF) exhibits a maximum value at theconcentration of approximately 3 mM of ammonium nickel sulfate when thesolution was left to stand for 16 hours or longer. Although the highestefficiency of nanoparticle formation (ECF) is exhibited when thesolution was left to stand for 16 hours, the yield of nanoparticleformation (YCF) under this condition is not 100% at any concentration ofammonium nickel sulfate, as is clear from FIGS. 8A, B, C and D. When theyield of nanoparticle formation (YCF) is not 100%, it turns out thatapoferritin which includes a nanoparticle and apoferritin which does notinclude apoferritin coexist, therefore, a further purification step forseparating these is required. Accordingly, it is believed that acondition of: the concentration of ammonium nickel sulfate of 3 mM; pHof approximately 8.58; and time period for leaving to stand the solutionpost preparation of 24 hours may be an optimal condition for producing anickel compound-apoferritin complex, in light of the efficiency offormation and the efficiency of purification.

Example 4 Test for Examining Influences of Ammonium Ion Upon Productionof Nickel Compound-Apoferritin Complex

[0125] In this Example, each of the solutions was first prepared, i.e.,anapoferritin solution in which HEPES buffer, CAPSO buffer andcommercially available apoferritin were dissolved, and an ammoniumnickel sulfate solution. Concentration and pH of each solution are asshown in Table 15. After preparing each solution, degassing of the HEPESbuffer and CAPSO buffer was immediately conducted. TABLE 15 Solution(pH) Concentration HEPES buffer (pH 7.5)  500 mM CAPSO buffer (pH 9.5) 500 mM Apoferritin solution  55 mg/ml Aqueous ammonia 1000 mM Nickelsalt solution  200 mM

[0126] Next, Milli Q water was provided, and carbon dioxide-bubbledwater was prepared by bubbling carbon dioxide into Milli Q water for 30minutes. Immediately thereafter, a reaction solution was prepared byadmixing each solution presented in Table 15 into the carbondioxide-bubbled water. In addition, a reaction solution was alsoprepared at the same time to which water which was subjected to merelydegassing without bubbling carbon dioxide (degassed water) was added instead of the carbon dioxide-bubbled water. The constitution of thereaction solution is presented in Table 16. TABLE 16 Solution (pH)Concentration HEPES  150 mM CAPSO  210 mM Apoferritin  0.3 mg/ml NH⁴⁺(supplied from aqueous ammonia)   20 mM Ammonium nickel sulfate solution  5 mM

[0127] In this Example, each reaction solution having the constitutionpresented in Table 16 was prepared to give the total volume of 3 ml,therefore, the amount added of each solution presented in Table 15 andcarbon dioxide-bubbled water or degassed water was as shown in Table 17.TABLE 17 Solution (pH) Concentration Carbon dioxide-bubbled water orAdded to give total degassed water volume of 3 ml HEPES buffer (500 mM,pH 7.5)   900 μl CAPSO buffer (500 mM, pH 9.5)  1260 μl Apoferritinsolution (55 mg/ml) 16.35 μl Aqueous ammonia   60 μl Ammonium nickelsulfate solution (200 mM)   75 μl

[0128] Each of thus resulting solutions was left to stand at 23° C. for24 hours. Thereafter, each reaction solution was centrifuged at 8000 Gfor 30 minutes. Each supernatant was collected, and the state of thesupernatant was observed.

[0129] Next, each of thus resulting supernatants was diluted to threefold with water, stained apoferritin with 2% gold glucose, and observedwith a transmission electron microscope (TEM) with fifty thousand foldmagnification. When staining is conducted with 2% gold glucose, entryinto the holding part in apoferritin does not take place. Therefore,apoferritin having a void holding part, and a nickelcompound-apoferritin complex can be discriminated.

[0130] Result of observation with a transmission electron microscope ofthe supernatant of the reaction solution in which carbon dioxide-bubbledwater was used is illustrated in FIG. 10A. As is shown in FIG. 10A,apoferritin including a nanoparticle of a nickel compound therein, withthe appearance of a protein portion having a white colored doughnut likeshape with a central portion which appears black, was observed.

[0131] The pH of the supernatant of the reaction solution in whichcarbon dioxide-bubbled water was used was further adjusted, and exploredfor the precipitation point. Consequently, the precipitation point ofNi(OH)₂ was pH of 8.65. Grounds for elevation of the pH of theprecipitation point compared to the aforementioned Examples 1 to 3 arebelieved to involve protection of a nickel ion (Ni²⁺) through theformation of a complex between the ammonium ion and the nickel ion(Ni²⁺). Range of pH, from the pH of 8.65 of this precipitation point tothe pH which is slightly lower than this pH of 8.65, is an optimal rangeof pH for forming a nanoparticle of the nickel compound. It could beascertained that aggregation of apoferritin was suppressed, and that ananoparticle of the nickel compound was formed in the holding part ofapoferritin, when the supernatant of the reaction solution in whichcarbon dioxide-bubbled water was used fell within the aforementionedoptimal range of pH.

[0132] When the pH of the supernatant of the reaction solution in whichcarbon dioxide-bubbled water is used rises higher than the pH of 8.65 ofthe precipitation point, apoferritin aggregates, and thus industrialutilization becomes difficult. Further, as the pH of the supernatant ofthe reaction solution in which carbon dioxide-bubbled water is usedlowers than the optimal range of pH for forming a nanoparticle,formation of nanoparticles declines, with no formation of nanoparticlesobserved at pH of 8.3.

[0133] On the other hand, a result of observation with a transmissionelectron microscope of the supernatant of the reaction solution in whichdegassed water was used is illustrated in FIG. 10B. As is shown in FIG.10B, apoferritin including a nanoparticle of a nickel compound therein,with the appearance of a protein portion having a white colored doughnutlike shape with a central portion which appears black, was not observed.Additionally, similar results were obtained even though the pH of thesupernatant of the reaction solution in which degassed water was usedwas variously altered. Although causes of failure of formation of ananoparticle are believed to involve influences of the ammonium ion, themechanism thereof is uncertain.

[0134] In any event, because the ammonium ion which is present in thereaction solution forms a complex with the nickel ion (Ni²⁺) to resultin stabilization, the pH of the precipitation point of Ni(OH)₂ becomeshigh in this Example. Importance of the presence of a carbonate ionand/or a hydrogen carbonate ion in the reaction solution for theformation of a nanoparticle, and suitability for forming thenanoparticle of keeping the pH of the reaction solution to be slightlylower than the pH of the precipitation point of Ni(OH)₂ are commonlyfound as in the aforementioned Examples 1 to 3.

Example 5 Test for Exploring Optimal Concentration of Ammonium Ion UponProduction of Nickel Compound-Apoferritin Complex

[0135] In this Example, each of the solutions was first prepared, i.e.,anapoferritinsolution in whichHEPESbuffer, CAPSO buffer and commerciallyavailable apoferritin (derived from equine spleen) were dissolved, andan ammonium nickel sulfate solution. Concentration and pH of eachsolution are as shown in Table 18. After preparing each solution,degassing of the HEPES buffer and CAPSO buffer was immediatelyconducted. TABLE 18 Solution (pH) Concentration HEPES buffer (pH 7.5) 500 mM CAPSO buffer (pH 9.5)  500 mM Apoferritin solution  55 mg/mlAqueous ammonia 1000 mM Ammonium nickel salt solution  200 mM

[0136] Next, Milli Q water was provided, and carbon dioxide-bubbledwater was prepared by bubbling carbon dioxide into Milli Q water for 30minutes. Immediately thereafter, each solution as shown in Table 18 wasadmixed into carbon dioxide-bubbled water to give the reaction solutionhaving the constitution presented in Table 19 with the total volume of 3ml. TABLE 19 Solution (pH) Concentration HEPES 150 mM CAPSO 150 to 300mM Apoferritin 0.3 mg/ml NH⁴⁺ (supplied from aqueous ammonia) 0, 10, 20,30 mM Ammonium nickel sulfate solution 5 mM

[0137] Each of the reaction solutions obtained as described above wasleft to stand at 23° C. for 48 hours. Thereafter, each reaction solutionwas centrifuged at 8000 G for 30 minutes. Each supernatant wascollected, and the state of the supernatant was observed.

[0138] Next, each of thus resulting supernatants was diluted to threefold with water, stained apoferritin with 2% gold glucose, and observedwith a transmission electron microscope (TEM) with fifty thousand foldmagnification. When staining is conducted with 2% gold glucose, entryinto the holding part in apoferritin does not take place. Therefore,apoferritin having a void holding part, and a nickelcompound-apoferritin complex can be discriminated.

[0139] Results of the above observation are illustrated in FIG. 11A foryield of nanoparticle formation (YCF), and in FIG. 11B for efficiency ofnanoparticle formation (ECF) versus pH of the supernatant of thereaction solution. The precipitation point of each solution was: pH of8.42 for the solution to which aqueous ammonia was not added, pH of 8.58at 10 mM of the ammonium ion (hereinafter, the ammonium ion in thisExample refers to one supplied from aqueous ammonia), pH of 8.65 at 20mMof the ammonium ion, andpH of approximately 8.92 at 30 mM of theammonium ion.

[0140] As is clear from the aforementioned results, the pH of theprecipitation point increases depending on the concentration of theammonium ion as added. Grounds for this event are believed to involveprotection of a nickel ion (Ni²⁺) through the formation of a complexbetween the ammonium ion and the nickel ion (Ni²⁺) as is also analyzedin Example 4. Range of pH, from the pH which is approximate to the pH ofthis precipitation point or slightly lower than this precipitation pointis optimal for forming nanoparticles in each of the solutions includingan ammonium ion at different concentrations. It could be ascertainedthat aggregation of apoferritin was suppressed, and a nanoparticle ofthe nickel compound was formed in the holding part of apoferritin whenthe supernatant of the reaction solution in which carbon dioxide-bubbledwater was used fell within the aforementioned optimal range of pH.

[0141] As is clear from FIG. 11B, the highest efficiency of nanoparticleformation (ECF) was exhibited by a solution including the aqueousammonia at the concentration of 20 mM, being 100% at the pH which isapproximate to the precipitation point. Having the efficiency ofnanoparticle formation (ECF) of 100% means that all apoferritin added tothe solution forms a nanoparticle in its holding part. In Example 3without the addition of aqueous ammonia, the efficiency of nanoparticleformation (ECF) did not reach to 100% under any condition (see, FIG. 9),therefore, it is revealed that efficient formation of nanoparticles wasenabled by adding an ammonium ion. In particular, in the constitution ofthis Example, the best yield of the nanoparticles is achieved when anammonium ion is added such that the concentration becomes 20 mM.Irrespective of the addition of aqueous ammonia, the yield ofnanoparticle formation (YCF) attained 100% at the pH which isapproximate to the precipitation point, respectively.

[0142] In this Example, high value of efficiency of nanoparticleformation (ECF) is obtained by adding an ammonium ion at an appropriateconcentration. In addition, importance of the presence of a carbonateion and/or a hydrogen carbonate ion in the reaction solution for theformation of a nanoparticle, and suitability for forming thenanoparticle of keeping the pH of the reaction solution to beapproximate to the pH of the precipitation point of Ni (OH)₂ or slightlylower than the same are commonly found as in the aforementioned Examples1 to 4.

Example 6 Production of Chromium Compound-Apoferritin Complex

[0143] In this Example, each of the solutions was first prepared, i.e.,an L chain apoferritin solution in which HEPES buffer, MES buffer andgenetically engineered apoferritin of L chain alone were dissolved, andan ammonium chromium sulfate solution. Concentration and pH of eachsolution are as shown in Table 20. After preparing each solution,degassing of the HEPES buffer and MES buffer was immediately conducted.TABLE 20 Solution (pH) Concentration HEPES buffer (pH 7.5) 500 mM MESbuffer (pH 5.5)  55 mM L chain apoferritin solution  55 mg/ml Ammoniumchromium sulfate solution 200 mM

[0144] Next, Milli Q water was provided, and carbon dioxide-bubbledwater was prepared by bubbling carbon dioxide into Milli Q water for 30minutes. Immediately thereafter, a reaction solution was prepared havingthe constitution presented in Table 21 by admixing each solutionpresented in Table 20 into the carbon dioxide-bubbled water. TABLE 21Solution (pH) Concentration HEPES  150 mM MES  210 mM L chainapoferritin  0.1 mg/ml Ammonium chromium sulfate   5 mM

[0145] In this Example, each reaction solution having the constitutionpresented in Table 21 was prepared to give the total volume of 3 ml,therefore, the amount added of carbon dioxide-bubbled water and eachsolution presented in Table 20 was as shown in Table 22. TABLE 22Solution (concentration, pH) Concentration Carbon dioxide-bubbled waterAdded to give total volume of 3 ml HEPES buffer (500 mM, pH 7.5)   900μl MES buffer (500 mM, pH 5.5)  1260 μl L chain apoferritin solution(27.5 mg/ml) 16.35 μl Ammonium chromium sulfate solution (200 mM)   75μl

[0146] The reaction solutions obtained as described above were left tostand at 23° C. for 24 hours. Thereafter, each reaction solution wascentrifuged at 8000 G for 30 minutes. Each supernatant was collected,and the state of the supernatant was observed.

[0147] Next, each of thus resulting supernatants was diluted to threefold with water, stained apoferritin with 2% gold glucose, and observedwith a transmission electron microscope (TEM) with fifty thousand foldmagnification. When staining is conducted with 2% gold glucose, entryinto the holding part in apoferritin does not take place. Therefore,apoferritin having a void holding part, and apoferritin including thenanoparticle of the chromium compound (i.e., chromiumcompound-apoferritin complex) therein can be discriminated.

[0148] Observation of each supernatant with a transmission electronmicroscope revealed a large number of apoferritin including ananoparticle of a chromium compound therein, with the appearance of aprotein portion having a white colored doughnut like shape, with acentral portion which appears black. Nanoparticles of the chromiumcompound had a spherical shape, with the diameter of 6 nm (standarddeviation: 1 nm). In other words, it is deemed that nanoparticles havinga uniform particle diameter could be obtained.

[0149] Because the pH of the reaction solution was adjusted to beapproximate to the precipitation point of Cr(OH)₂, the supernatant ofthe reaction solution was slightly turbid. When the supernatant wasobserved with a transmission electron microscope, apoferritin includinga nanoparticle of a chromium compound therein, with the appearance of aprotein portion having a white colored doughnut like shape, with acentral portion which appears black was observed.

[0150] Under the condition employed in this Example, nanoparticles ofthe chromium compound were ascertained within about 50% of apoferritinin the supernatant. In other words, the yield of nanoparticle formation(YCF) was about 50%. Although production of nanoparticles of thechromium compound was attempted with the reaction solution under thesame condition except that degassed water was used in stead of carbondioxide-bubbled water, formation of nanoparticles of the chromiumcompound was not ascertained.

Example 7 Production of Copper Compound-Apoferritin Complex

[0151] In this Example, each of the solutions was first prepared, i.e.,an apoferritin solution in which MES buffer, and commercially availableapoferritin (derived from equine spleen) were dissolved, and an ammoniumcopper sulfate solution. Concentration and pH of each solution are asshown in Table 23. After preparing each solution, degassing of the MESbuffer was immediately conducted. TABLE 23 Solution (pH) ConcentrationMES buffer (pH 6.0) 500 mM Apoferritin solution  5 mg/ml Ammonium coppersulfate solution  10 mM

[0152] Next, Milli Q water was provided, and carbon dioxide-bubbledwater was prepared by bubbling carbon dioxide into Milli Q water for 30minutes. Immediately thereafter, a reaction solution was prepared havingthe constitution presented in Table 24 by admixing each solutionpresented in Table 23 into the carbon dioxide-bubbled water. TABLE 24Solution (pH) Concentration MES  100 mM Apoferritin 0.15 mg/ml Ammoniumcopper sulfate  1.2 mM

[0153] In this Example, each reaction solution having the constitutionpresented in Table 23 was prepared to give the total volume of 3 ml,therefore, the amount added of carbon dioxide-bubbled water and eachsolution presented in Table 23 was as shown in Table 25. TABLE 25Solution (concentration, pH) Concentration Carbon dioxide-bubbled waterAdded to give total volume of 3 ml MES buffer (500 mM, pH 6.0) 600 μlApoferritin solution (5 mg/ml)  90 μl Ammonium copper sulfate solution(10 mM) 360 μl

[0154] The reaction solutions obtained as described above were left tostand at 23° C. for 24 hours. Thereafter, each reaction solution wascentrifuged at 8000 G for 30 minutes. Each supernatant was collected,and the state of the supernatant was observed.

[0155] Next, each of thus resulting supernatants was diluted to threefold with water, stained apoferritin with 2% gold glucose, and observedwith a transmission electron microscope (TEM) with fifty thousand foldmagnification. When staining is conducted with 2% gold glucose, entryinto the holding part in apoferritin does not take place. Therefore,apoferritin having a void holding part, and apoferritin including thenanoparticle of the copper compound (i.e., copper compound-apoferritincomplex) therein can be discriminated.

[0156] Observation of each supernatant with a transmission electronmicroscope revealed a large number of apoferritin including ananoparticle of a copper compound therein, with the appearance of aprotein portion having a white colored doughnut like shape, with acentral portion which appears black. Nanoparticles of the coppercompound (Cu(OH)₂) had a spherical shape, with the diameter of 6 nm(standard deviation: 1 nm). In other words, it is deemed thatnanoparticles having a uniform particle diameter could be obtained.

[0157] Because the pH of the reaction solution was adjusted to beapproximate to the precipitation point of the copper compound (Cu(OH)₂),the supernatant of the reaction solution was slightly turbid. When thesupernatant was observed with a transmission electron microscope,apoferritin including a nanoparticle of a copper compound therein, withthe appearance of a protein portion having a white colored doughnut likeshape, with a central portion which appears black was observed.

[0158] Under the condition employed in this Example, nanoparticles ofthe copper compound were ascertained within about 30 to 40% ofapoferritin in the supernatant. In other words, the yield ofnanoparticle formation (YCF) was about 30 to 40%.

[0159] As explained hereinabove, nanoparticles having a uniform particlediameter can be obtained according to the present invention. Moreover,according to the present invention, to obtain a compound particle isenabled in a cavity part of a protein also with a metal ion which hadnot been hitherto reported on the formation of a particle in the cavitypart of a protein, and in addition, nanoparticles having a uniformparticle diameter composed of such a metal ion can be obtained.

[0160] According to the present invention, nanoparticles having auniform particle diameter can be provided. Nanoparticles which areprovided by the present invention can be utilized for semiconductorchips, single-gun devices, quantum dots, light emitting elements and thelike through utilizing the quantum effects of the same.

[0161] Furthermore, the nanoparticles provided by the present inventioncan be utilized as a material with a sophisticated function, highperformance, high density and high precision, such as e.g., opticalfunction coating materials, electromagnetic wave shielding materials,materials for secondary batteries, fluorescent materials, materials forelectronic parts, materials for magnetic recording, materials forgrinding, materials for cosmetics and the like.

[0162] It will be obvious to those having skill in the art that manychanges may be made in the above-described details of the preferredembodiments of the present invention. The scope of the presentinvention, therefore, should be determined by the following claims.

What is claimed is:
 1. A method of the production of a nanoparticlewhich comprises a step of forming a nanoparticle including a compound ofa metal ion in a cavity part of a protein, in a solution containing theprotein having the cavity part therein, the metal ion, and a carbonateion and/or a hydrogen carbonate ion.
 2. The method of the production ofa nanoparticle according to claim 1, wherein said compound is ahydroxide.
 3. The method of the production of a nanoparticle accordingto claim 2, wherein said metal ion is any one of a nickel ion (Ni²⁺), achromium ion (Cr²⁺) or a copper ion (Cu²⁺).
 4. The method of theproduction of a nanoparticle according to claim 3, wherein said metalion is a nickel ion.
 5. The method of the production of a nanoparticleaccording to claim 3, wherein said metal ion is a chromium ion.
 6. Themethod of the production of a nanoparticle according to claim 3, whereinsaid metal ion is a copper ion.
 7. The method of the production of ananoparticle according to claim 2, wherein pH of said solution isapproximately equal to a precipitation point of a hydroxide of saidmetal ion.
 8. The method of the production of a nanoparticle accordingto claim 4, wherein pH of said solution is 8 or greater and 9 or less.9. The method of the production of a nanoparticle according to claim 4,wherein said solution further comprises an ammonium ion.
 10. The methodof the production of a nanoparticle according to claim 9, wherein pH ofsaid solution is greater than 8.3 and equal to or less than 8.65. 11.The method of the production of a nanoparticle according to claim 1,wherein said protein is at least one of apoferritin, Dps protein, CCMVprotein, TMV protein or a heat shock protein.
 12. The method of theproduction of a nanoparticle according to claim 1, wherein said solutioncomprises a carbonate ion and/or a hydrogen carbonate ion produced bybubbling carbon dioxide thereto.
 13. The method of the production of ananoparticle according to claim 1 further comprise a step of eliminatingthe protein by a heat treatment after forming said nanoparticle.
 14. Ananoparticle including a compound of a metal ion, which is formed in acavity part of a protein, in a solution containing the protein havingthe cavity part therein, the metal ion, and a carbonate ion and/or ahydrogen carbonate ion.
 15. A complex comprising a protein having acavity part therein, and a nanoparticle formed in the cavity part of theprotein; the nanoparticle being a nanoparticle comprising a compound ofa metal ion, which is formed in the cavity part of the protein, in asolution containing the protein, the metal ion, and a carbonate ionand/or a hydrogen carbonate ion.